Analysis of drugs of abuse in biological matrix using Time of Flight technology Niclas Stephanson, PhD Department of Clinical Pharmacology Karolinska University Hospital Karolinska Institutet Stockholm, Sweden
Chromatographic screening Needed when no immunochemical assay is available - For example designer drugs, internet drugs Needed to reduce false positive/negative
Aims of the project Multicomponent method for target and unknown compounds Sensitivity 100% and specificity 99% All positive results to be confirmed Complement to immunochemical screening Alternative to immunochemical screening
Project: LC-TOF Screening of randomly selected urine samples (N=812) Immunochemical technique LC TOF (UPLC - Xevo QTOF, R: ~ 10 000) Confirmation and quantification UPLC MS/MS (MRM) LC MS (SIM)
% Initial results Screening of 32 drugs of abuse with a wide range of polarities Direct injection of diluted urine (1:10) Screening of 10 drugs of abuse Diluted urine (1:5) bland 33 opt 114 (HSS) 1/10 urin gscren_090330_8 100 M6G Morphine Ketobemidone Mephedrone 2.33 1: MRM of 13 Channels ES+ 340.58 > 266.3 (Propoxyfen) 9.73e5 Propoxyphene Norpropoxyphne 0 Time 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00
LC TOF: Analytes Amphetamines (IS: Amphetamine-d5) Amphetamine Methamphetamine Opiates Morphine-3-glucuronide Morphine-6-glucuronide Morphine Codeine Codeine-6-glucuronide 6-MAM (excluded from project) Opioids (IS: MDMA-d4) Buprenorphine Buprenorphine glucuronide
% LC TOF: Summary of the method features Chromatographic conditions: - Injection of diluted urine (1:5) - C18, HSS-T3 UPLC-column - Gradient elution Mobile phase: 0.1% formic acid/acetonitrile - Flow: 1 ml/min - Injection volume 2 µl TOF/MS: ES +, scan: 100 1000 m/z Scan time 0.08 sec, cone 20 V DRE used for every second scan LockSpray: 1 point m/z 556.2771 (LeuEnk) Total analysis time: 4 min. 100 1 2;3 4 5 6 7 8 9 1. M3G: RT: 0.31 2. M6G: RT: 0.46 3. Morphine: RT: 0.46 4. C6G: RT: 0.68 5. Codeine: RT: 0.75 6. Amphetamine: RT: 0.78 7. Methamphetamine: RT: 0.89 8. BuprG: RT: 1.98 9. Buprenorphine: RT: 2.20 0 Time 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30
Method validation Linearity: r 2 0.97 (n=10, conc. levels 100 10000 ng/ml; Buprenorphine/buprGl: conc. 10 1000 ng/ml) Method imprecision (RSD): within-run and between-run < 20% Selectivity: No interferences observered Carry over: < 0.01% LOD: Buprenorphine: 2.0 ng/ml Buprenorphine Glucuronide: 2.6 ng/ml Other analytes: < 10 ng/ml
Method validation: Evaluation of matrix effect Post column infusion of water solutions of analytes into MS with simultaneous injection of urine matrix The infusion chromatogram of analytes is overlaid with the M3G and M6G calibrator chromatogram
Identification criteria MassLynx ChromaLynx: Rt tolerance 0.05 min Absolute mass accuracy ± 0.01 Da Tentative within ± 0.01 0.02 Da TargetLynx (quantification) Chromatogram mass window 0.05 Da Rt tolerance ± 0.05 min
Identification criteria (chromalynx)
TargetLynx quantification connection to Chromalynx
Results Cutoff limits (ng/ml) Parameter Immunoassay (CEDIA) TOF screening Confirmation LC-MS Amphetamines 500 100 100 Opiates 300 100 100 Buprenorphines (total) 5 5 5
Results Comparison of performance for CEDIA immunoassay and LC-TOF screening Amphetamines n=812 Opiates n=779 Buprenorphines n=796 CEDIA LC-TOF CEDIA LC-TOF CEDIA LC-TOF Positive rate 26.8% 28.4% 26.6% 26.1% 28.1% 27.3% False positive rate 7.3% 0%* 5.8% 0%** 6.7% 3.2% False negative rate 4.4% 1.8% 12.9% 0.6% 0.5% 0% Agreement 94.2% 97.4% 97.4% * All samples contained amphetamine **All samples contained M3G and/or CG
Results - Performance of the LC-TOF screening compared with confirmation results Compound (number of samples) M3G (n=779) M6G (n=779) Morphine (n=779) CG (n=779) Codeine (n=779) Amphetamine (n=812) Methamphetamine (n=812) Buprenorphine (n=796) BG (n=796) False positives Detected false negatives Relative rate Number of samples Number of samples 0% 0 0 6.6% 8 6 4.8% 3 1 0% 0 2 2.9% 2 1 0% 0 1 8.0% 7 1 1.8% 4 0 2.3% 5 0
Intensity (%) Example: chromatogram from authentic patient sample BG: m/z 644.3435 1.98 0 0.40 0.80 1.20 1.60 2.00 2.40 2.80 Buprenorphine: m/z 468.3114 2.20 0 0.40 0.80 1.20 1.60 2.00 2.40 2.80 Time
Difficulties Isotopic contributions for 3-deuterated (D 3 ) IS M3G+3 (3 of C13):C23H27NO9; MW=464.1786; M3G-D 3 : C23H30NO9; MW=464.1921 Difference: ~ 0.013 Da M3G+3 (3 of C13) contributes with 0.25% for A+3 channel: affects mass accuracy of M3G-D 3 M3G: Conc 2.5 µg/ml
Difficulties Matrix interferences on 6MAM peak 6MAM: C19H21NO4; (M +H) + =328.1549 Interference: (M +H) + =328.1617 Difference: 0.007 Da To separate: needed resolution: ~ 50 000 6MAM: 4 ng/ml Blank urine 6MAM: 4 ng/ml Blank urine
Difficulties - Interferences on 6MAM Interference in the method affects linearity and mass accuracy of 6MAM Calibration curve for 6MAM with conc: 1.25 ng/ml, 3.75 ng/ml and 30 ng/ml Blank urine 6MAM calibration curve IS
Review results 57% of all samples had one or more tentative finding Almost all of them were true negatives
Conclusions: TOF vs Immunoassay TOF screening sensitivity higher than immunoassay for chosen compounds TOF screening specificity very high Chromatographic separation of interferences with similar masses and high resolving power of instrument is of great importance Limited number of analytes could be analyzed with this TOF-procedure. TOF screening is more time consuming than immunoassay (more than double)
Acknowledgements Olof Beck, PhD Aljona Salem Per Åke Hynning Martin Josefsson, PhD Tomas Villén